Dactylfungins and Tetralones: Bioactive Metabolites from a Nematode-Associated Laburnicola nematophila

A chemical investigation of Laburnicola nematophila, isolated from cysts of the plant parasitic nematode Heterodera filipjevi, affored three dactylfungin derivatives (1–3) and three tetralone congeners (4–6). Dactylfungin C (1), laburnicolin (4), and laburnicolenone (5) are previously undescribed natural products. Chemical structures of the isolated compounds were determined based on 1D and 2D NMR spectroscopic analyses together with HR-ESI-MS spectrometry and comparison with data reported in the literature. The relative configurations of compounds 1, 2, and 4–6 were determined based on their ROESY data and analysis of their coupling constants (J values). The absolute configurations of 4–6 were determined through the comparison of their measured and calculated TDDFT-ECD spectra. Compounds 1–3 were active against azole-resistant Aspergillus fumigatus.

F ungal natural products have emerged as a prolific source of novel antimicrobial drugs and therapeutic agents. 1−3 While the challenge of multiresistant bacteria has been extensively acknowledged, the issue of multidrug-resistant pathogenic fungi is frequently overlooked. 4This oversight is particularly noteworthy considering the limited availability of compound classes suitable for treating invasive fungal infections. 3Recognizing the gravity of the situation, the World Health Organization (WHO) has designated Cryptococcus neoformans and Aspergillus fumigatus alongside Candida spp. in the critical priority group. 5Cryptococcosis caused by C. neoformans, in particular, is a crucial concern for immunocompromised individuals such as those with an HIV infection. 6imilarly, A. f umigatus is also a major threat for immunosuppressed patients. 7,8The emergence of azole-resistant strains, in particular those belonging to A. f umigatus, has become a real threat and underscores the critical need to identify novel drug targets. 6,9he genus Laburnicola was introduced by Wanasinghe et al. 10 and belongs to the Pleosporales, the largest within the Dothideomycetes. 11The genus shows a broad spectrum of potential hosts and is described as an endophyte and a nematode antagonist. 12−15 In this report, we chemically explored brown rice based solid-state and liquid YM 6.3 cultures of Laburnicola nematophila 20AD (DSM 112866) and K01 (DSM 112867) strains, isolated from eggs of the cereal cyst nematode Heterodera f ilipjevi. 12The present study deals with the evaluation of the secondary metabolites of L. nematophila, some of which revealed significant antifungal activity against human pathogenic azole-resistant strains.and the polyalcohol moiety to be attached at C-2 and C-5 of the α-pyrone functionality, respectively.The long side-chain in 1 featured two pairs of olefinic protons at δ H 5.58 (H-3″)/δ H 6.29 (H-4″) and δ H 5.55 (H-11″)/δ H 6.04 (H-12″), as elucidated from the 1 H NMR (Table 1) and 1 H− 1 H COSY spectra (Figure 1), which were deduced to be in trans configuration based on their large coupling constants (J values ≥ 15.0−16.0Hz).

Journal of Natural Products
obtained and by comparison with the reported spectral data of dactylfungins, 16,20 compound 1 was identified as a previously undescribed derivative that was trivially named dactylfungin C.
Compound 2 was obtained as a yellow amorphous solid.Its molecular formula was determined to be C 38 H 60 O 9 based on its HR-ESI-MS data, indicating nine degrees of unsaturation similar to 1. Based on the molecular formulas, compound 2 was suggested to be a deoxygenated derivative of 1. Expectedly, both 1 H and 13 C NMR data of 1 and 2 (Table 1) revealed a close coherence apart from one main difference due to the disappearance of one methine sp 3 carbon at δ C 79.0 (C-16″) and the presence of a secondary sp 3 carbon at δ C 31.31 (C-16″) that was correlated via the HSQC spectrum (Figure S15) to diastereotopic methylene protons at δ H 1.08/δ H 1.29 ppm.In the 1 H− 1 H COSY spectrum (Figures 1, S13), the previous diastereotopic methylene protons featured a spin-system extending over one aliphatic methine proton at δ H 1.26 (m, H-17″) to a second diastereotopic methylene group at δ H 1.15/δ H 1.29 (H 2 -18″) ending with a terminal triplet methyl group at δ H 0.86 (t, J = 7.3 Hz, H 3 -19″).The results obtained suggest that compound 2 is a 16-deoxy derivative of 1.A literature search of 2 revealed that a related derivative was patented in 2005 as a synthetic anti-inflammatory lactone. 21urther confirmation of the structure of 2 was obtained via 2D NMR spectra including 1 H− 1 H COSY and HMBC (Figure 1) spectra, which revealed similar correlations to those shown for 1.Similar to 1, the relative configuration at the chiral centers in 2 on both the polyalcohol residue (Figure 1a) and the aliphatic side-chain was determined based on the analysis of the coupling constants (J values) and the ROESY spectrum (Figures 1, S16) taking into consideration a common biosynthetic origin.Accordingly, the relative configuration of 2 was suggested to be (1′R*,2′S*,3′R*,5′R*,2″S*,7″S*,8″S*,-9″S*,15″R*,17″S*).The absolute configuration of 2 necessitates applying the scheme previously described, 18,19 which was not possible due to the limited amount obtained (1.3 mg).According to the obtained spectral data of 2, it was identified as a 16-deoxy derivative of 1 and it was given the trivial name dactylfungin D.
The ROESY spectrum of 4 (Figures 3, S30) further confirmed the depicted structure by revealing key ROE correlations from both H-4 and OCH 3 -6 to the aromatic proton at δ H 6.71 (d, J = 0.9 Hz) assigned to H-5.The absolute configuration of the carbinol group at C-4 was predicted to be (R) based on biosynthetic considerations and the assigned absolute configurations of the co-isolated compounds 5 and 6.When comparing the experimental ECD spectra of 4, 5, and 6, it was evident that 4 has a much smaller intensity due to the presence of two interacting similar aromatic chromophores connected with a methylene linker, where the different conformer groups with comparable populations can cancel the ECD contributions of one another. 26The Boltzmann distribution has a fundamental effect on the spectrum that can be challenging to estimate precisely due to the known limitations of DFT functionals. 27According to the obtained results and by comparison to the reported literature, 22−25 compound 4 was identified as a previously undescribed heterodimer compound and was named laburnicolin.
Being a heterodimer composed of an isocoumarin and a tetralone subunit, compound 4 was probably biosynthesized through an acetate−malonate pathway influenced by polyketide synthase (PKS).A plausible biosynthetic scheme of laburnicolin (4) (Figure 4) suggests that its biosynthesis is via two parallel phases of a PKS pathway to afford the building subunits 10-norparvulenone (6) and 2-methyl-5,6,8-trihydroxyisocoumarin, which would be finally combined via nucleophilic condensation to yield laburnicolin (4).
Compound 5 was isolated as a white amorphous solid.Its molecular formula was determined to be C 15 H 16 O 5 based on its HR-ESI-MS data, indicating eight degrees of unsaturation.The 1 H and 13 C NMR spectral data of 5 (Table 2) revealed comparable resonance values to those measured and reported for 10-norparvulenone (6). 22A detailed investigation of the 1 H and 13 C NMR data of 5 (Table 2) revealed the presence of a second ketocarbonyl carbon at δ C 202.8 (C-11) and two olefinic carbon atoms at δ C 135.1 (C-9) and δ C 130.6 (C-10) directly correlated via HSQC to two olefinic protons at δ H 7.98 and δ H 7.25 appearing as doublet signals with a coupling constant of 16.5 Hz, indicating a trans configuration.In addition, the 1 H NMR spectrum of 1 (Table 2) revealed a singlet methyl group at δ H 2.35 (H 3 -12) that exhibited together with H-9 and H-10 common key HMBC correlations (Figure 3) to the ketocarbonyl carbon assigned to C-11.By comparing the obtained 1 H/ 13 C NMR data of 5 (Table 2) and 10- norparvulenone (6) (Li et al., 2010), 22 it was concluded that 5 features an α,β-unsaturated ketone moiety replacing the hydroxymethylene functionality in 10-norparvulenone (6).
To confirm the position of the α,β-unsaturated ketone moiety on the tetralone core, its HMBC spectrum (Figure 3) revealed further key correlations from H-10 and H-5 (δ H 6.89, s) to an unprotonated olefinic carbon at δ C 110.7 (C-7), whereas other key HMBC correlations were recognized from H-9 to two oxygenated aromatic carbons at δ C 165.6 (C-8) and 166.5 (C-6).These key HMBC correlations confirmed the binding of the α,β-unsaturated ketone moiety at C-7 in 5.The ROESY spectrum of 5 (Figure 3) revealed similar key ROE correlations to those exhibited by 4 from both H-4 (δ H 4.83, dd, J = 9.4, 4.1 Hz) and OCH 3 -6 (δ H 4.04, s) to the aromatic proton at δ H 6.89 assigned to H-5.To determine the absolute configuration, the TDDFT-ECD method was applied on the (R) enantiomer of 5. 28,29 The Merck molecular force field (MMFF) conformational search yielded 57 conformer clusters in a 21 kJ mol −1 energy window, the ωB97X/TZVP PCM/ MeOH reoptimization of which resulted in 16 low-energy conformers over 1% Boltzmann population.The computed ECD spectra at various levels of theory gave a good agreement with the experimental ECD spectrum, underestimating only the positive shoulder at 241 nm, and it suggested the (R) absolute configuration (Figure 5).
For tetralones, a semiempirical ECD helicity rule 30,31 may be applied to correlate the n−π* transition of the conjugating carbonyl group above 300 nm with the helicity of the carbocyclic ring and hence the absolute configuration.According to the literature data, some substituted tetralones with M helicity of the fused carbocyclic ring produced a positive n−π* CE (Cotton effect), 32−35 while others with P helicity also showed positive n−π* CEs. 36,37The computed conformers of (R)-5 were classified into two groups (Figure 6).
On the basis of the P and M helicity of the carbocyclic ring, which gave nearly opposite ECD spectra (Figure S53) and as suggested by the Kohn−Sham orbitals, the carbonyl n−π* CE was identified as the fourth transition with negative sign, which was derived from M helicity of the high population conformers (Figure S39).Furthermore, in the experimental ECD spectrum there is only a slightly visible shoulder at 303 nm corresponding to this transition that makes application of the helicity rule ambiguous for similar derivatives.Based on the obtained results, compound 5 was identified as a previously undescribed tetralone derivative, and it was given the trivial name laburnicolenone.
In the literature, the (R) absolute configuration was reported for 6 on the basis of the opposite specific optical rotation to that of O-methylasparvenone. 38 Although structurally related derivatives often exhibit the same sign of optical rotation, it is known that even slight structural modifications in the substitution pattern may invert the sign of the specific optical rotation. 39,40In order to elucidate the absolute configuration of 6 independently, ECD calculations were carried out.DFT reoptimization of the 33 initial MMFF conformers of (R)-6 resulted in 16 low-energy conformers, the ECD calculations of which were performed at various levels of theory, affording excellent agreement with the experimental ECD spectrum (Figure 7).Similarly to 5, the low-energy computed conformers of 6 could be classified into two groups with nearly mirror-image ECD spectra and opposite helicity, where the first group with M helicity was more abundant than the second with P helicity (Figures 8 and S54).
By checking the Kohn−Sham orbitals (Figure S52), the n−π* transition was identified as the second ECD transition with a positive sign, which appeared in the experimental ECD spectrum (Figure 7) below 300 nm as the second CE.This can be a source of confusion when the semiempirical tetralone   helicity rule would be applied for similar scaffolds, and thus ECD calculations are recommended.The TDDFT-ECD calculations confirmed that the carbocyclic ring adopted preferably M helicity in both (R)-5 and (R)-6 with an equatorial 4-OH group, while their n−π* CE was found to have opposite signs: negative for 5 and positive for 6.
Biological Assays.The effects of all isolated metabolites are shown in Table 3 (complete data set in Table S1).Besides the assessment of antimicrobial and cytotoxic activities, dactylfungins (1−3) were evaluated in an in-depth assay for their antifungal activity against different strains of Aspergillus f umigatus including azole-resistant strains, as well as Cryptococcus neoformans and Mucor plumbeus.The evaluation demonstrated significant antifungal activity for all three substances (1−3).Dactylfungin D (2) showed overall the highest potency against the tested organisms, especially against A. f umigatus as well as azole-resistant strains and moderate activity against C. neoformans and Rhodotorula glutinis.YM-202204 (3) revealed activity across multiple fungal species including higher activity against A. f umigatus (azole-resistant) and moderate activities against C. albicans and A. f umigatus, while its highest activity was against M. plumbeus.Dactylfungin C (1) displayed high impact only on the growth of A. f umigatus and weak activity on its azole-resistant strain.Apart from their weak cytotoxic effects, compounds 4 and 5 revealed no other biological activity in the conducted assays, whereas 10-norparvulelone (6) illustrated no activity against the tested organisms or cell lines.None of the tested compounds (1, 2, 4−6) exhibited significant activity against Caenorhabditis elegans (Table S2).In this study, dactylfungin derivatives (1−3), featuring an α-pyrone motif substituted with a polyacohol and a long aliphatic side-chain, were interesting with regard to the impact of side-chain substitution pattern on their antifungal activities.In particular, the higher hydrophilicity attained by introducing an additional hydroxy group in 1 compared to 2 seems to negatively affect its antifungal activity especially against azole-resistant A. f umigatus strains.This observation is supported by the study of Charria-Giroń et al., 20 which showed a decreased efficacy of hydroxylated derivative 21″-hydroxy-dactylfungin A compared to dactylfungin A produced by Amesia hispanica.
Laburnicola nematophila, a member of the family Didymosphaeriaceae within the Massarineae, exhibits a fascinating parasitic interaction with nematode eggs. 12,41This study identified L. nematophila as a novel producer of dactylfungins, potent antifungal agents that strongly inhibit the growth of A. f umigatus including azole-resistant strains.The hydroxylation pattern of the hydrophobic aliphatic side-chain significantly affected their activities.The described tetralones showed no biological activity other than their weak cytotoxic effects.Further research investigating their metabolism and interaction in their ecosystems could improve the knowledge of their corresponding ecological roles and would allow applications as an antibiotic producer or as a biocontrol agent.
Fungal Material and Identification.The strains of Laburnicola nematophila 20AD (DSM 112866) and K01 (DSM 112867) were isolated from the eggs of the cereal cyst nematode Heterodera f ilipjevi
Cultivation and Metabolite Extraction.The seed cultures, containing 200 mL of Q6/2 medium (D-glucose 2.5 g L −1 , glycerol 10 g L −1 , cottonseed flour 5 g L −1 , pH 7.2) in a 500 mL Erlenmeyer flask, were inoculated with 5 × 25 mm 2 sections of mycelium grown on YM 6.3 agar and cultivated at 23 °C and shaking at 140 rpm in the dark.After reaching sufficient biomass, the culture broth was homogenized using an Ultra-Turrax (T25 easy clean digital, IKA) equipped with an S25 N-25F dispersing tool at 10,000 rpm for 10 s.This seed culture served as the inoculum for subsequent cultivations in BRFT (K 2 HPO 4 : 0.5 g L −1 ; sodium tartrate: 0.5 g L −1 ; yeast extract: 1 g L −1 ; 100 mL of solution was added to 28 g of brown rice and autoclaved) and inoculated with 6 mL of the YM 6.3 media seed culture.
Solid-State Fermentation.For the strain 20AD (DSM 112866), 20 Erlenmeyer flasks containing BRFT media were prepared and inoculated with 6 mL of homogenized seed culture each.Subsequently, 12 flasks were cultivated for 4 weeks, and 8 flasks were cultivated for 6 weeks in the dark at room temperature.After incubation, each culture flask was harvested by adding 3 × 250 mL of acetone, mixed, and extracted, following the previously described protocol. 15The total extracts were then defatted by liquid−liquid fractionation between n-heptane and methanol.Both fractions (nheptane and methanol) were evaporated to dryness and analyzed with HPLC-DAD-MS.Thirty-five and 15 flasks were prepared for strain K01 (DSM 112867) using the same parameters outlined above.
BRFT cultivation of K01 (DSM112867) resulted in a methanol extract of 3.8 g, which was fractionated by a Grace Reveleris X2 flash chromatography system utilizing a FlashPure ID silica 40 g cartridge and a flow rate of 40 mL min −1 , using parameters outlined above.The fractions from 3.5 to 15.6 min were combined and dried under reduced pressure, yielding a solid residue (396 mg), which was divided into three parts and further processed with a Gilson PLC 2250 system equipped with a Gemini C 18 column (250 × 50 mm, 10 μm, Phenomenex, Aschaffenburg, Germany); the mobile phase and the applied gradient were as follows: solvent A (H 2 O + 0.1% FA), solvent B (MeCN + 0.1% FA), flow rate: 40 mL min −1 , gradient: starting at 5% B for 5 min, progressing to 80% B in 50 min, reaching 100% B after additional 10 minutes, and keeping the 100% B for 10 min.The collected fractions from 42.4 to 42.9 min were pooled and dried in vacuum, affording a solid residue (25 mg).For the isolation of compound 5 (4.2 mg, t R = 44.5−47.0min), the latter residue ( Antimicrobial Assay.The antimicrobial activity was determined by a serial dilution assay to assess the minimum inhibitory concentration (MIC) of the isolated metabolites against different Gram-positive (Bacillus subtilis, Mycolicibacterium smegmatis, and Staphylococcus aureus) and Gram-negative (Acinetobacter baumannii, Chromobacterium violaceum, Escherichia coli, and Pseudomonas aeruginosa) bacteria and against five fungi (Candida albicans, Mucor hiemalis, Rhodotorula glutinis, Schizosaccharomyces pombe, and Wickerhamomyces anomalus), applying the same methods as previously described. 20A 20 μL amount of methanol was used as a negative control, and positive controls were selected based on the tested organism.Oxytetracycline, ciprobay, kanamycin, and gentamicin were used as controls for B. subtilis, A. baumannii, M. smegmatis, and P. aeruginosa, respectively, and nystatin was used for all fungal strains.In-depth analysis of the antifungal activity was performed as described by S ̌tepańek et al. 42 in a serial dilution assay.Methanol was used as a negative and amphotericin B as a positive control.The fungal strains, which are important human pathogens, were incubated in malt extract broth (Oxoid, Basingstoke, UK) for filamentous fungi and in YM 6.3 for yeasts in combination with the metabolites (1−3) in a concentration range of 100−0.13,33−0.13, and 16.7−0.013μg mL −1 .After 12 h (C.albicans), 48 h (A.f umigatus CCF 3522, Cr. neoformans, and M. plumbeus), and 72 h (others) the MIC was determined according to the published guidelines. 43All MIC values were determined in biological duplicates.
Nematicidal Assay.Nematicidal effects were assessed with Caenorhabditis elegans in a 48-well flat-bottom plate.Metabolites 1, 2, and 4−6 were tested at concentrations of 100, 50, and 10 μg mL −1 in biological triplicates.Ivermectin was used as a positive control at a concentration of 1 μg mL −1 , and methanol as a negative control.The assay was performed as described by Phutthacharoen et al. 44 Computational Section.Mixed torsional/low-mode conformational searches were carried out by means of the Macromodel 10.8.011 software using the MMFF with an implicit solvent model for CHCl 3 and applying a 21 kJ mol −1 energy window. 45Geometry reoptimizations of the resultant conformers (ωB97X/TZVP PCM/ MeOH) and TDDFT-ECD (B3LYP/TZVP PCM/MeOH, BH&HLYP/TZVP PCM/MeOH, CAM-B3LYP/TZVP PCM/ MeOH, and PBE0/TZVP PCM/MeOH) calculations were performed with the Gaussian 16 package. 46ECD spectra were generated as sums of Gaussians with a 4200 cm −1 width at half-height, using dipole-velocity-computed rotational strength values. 47Boltzmann distributions were estimated from the DFT energies.Visualization of the results was performed by the MOLEKEL 5.4 software package. 48ASSOCIATED CONTENT

a
Measured in DMSO-d 6 at 125 (for 13 C) MHz.b Measured in DMSO-d 6 at 500 (for 1 H) MHz.c Assignment confirmed by HMBC and HSQC spectra.d Measured in methanol-d 4 at 125 MHz e Measured in methanol-d 4 at 500 MHz.

Figure 5 .
Figure 5. Experimental ECD spectrum of 5 in MeOH compared with the BH&HLYP/TZVP PCM/MeOH ECD spectrum of (R)-5 computed for the low-energy ωB97X/TZVP PCM/MeOH conformers.The bars represent the rotational strength values of the lowest-energy conformer.

Figure 6 .
Figure 6.Overlapped geometries for two groups of the low-energy ωB97X/TZVP PCM/MeOH conformers of (R)-5: group A with M helicity and an equatorial hydroxy group represented by conformers A−H, and group B with P helicity and an axial hydroxy group represented by conformers I−P.

Figure 7 .
Figure 7. Experimental ECD spectrum of 6 measured in MeOH compared with the CAM-B3LYP/TZVP PCM/MeOH ECD spectrum of (R)-6 computed for the low-energy ωB97X/TZVP PCM/MeOH conformers.The bars represent the rotational strength values of the lowest-energy conformer.

Figure 8 .
Figure 8. Two groups of the low-energy ωB97X/TZVP PCM/MeOH conformers of (R)-6: group A with M helicity and an equatorial hydroxy group represented by conformers A−H, and group B with P helicity and an axial hydroxy group represented by conformers I−P.